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Abstract

Molecular machines embedded in a Langmuir monolayer at the air-water interface can
be operated by application of lateral pressure. As part of the challenge associated
with versatile sensing of biologically important substances, we here demonstrate discrimination
of nucleotides by applying a cholesterol-armed-triazacyclononane host molecule. This
molecular machine can discriminate ribonucleotides based on a twofold to tenfold difference
in binding constants under optimized conditions including accompanying ions in the
subphase and lateral surface pressures of its Langmuir monolayer. The concept of mechanical
tuning of the host structure for optimization of molecular recognition should become
a novel methodology in bio-related nanotechnology as an alternative to traditional
strategies based on increasingly complex and inconvenient molecular design strategies.

Introduction

Supramolecular structures constructed through bottom-up processes play crucial roles
in nanoscience and nanotechnology [1,2]. In particular, those structures can be applied in bio-related nanotechnologies such
as drug discrimination. Molecular assemblies immobilized at the air-water interface
are appropriate media for incorporation of the sensing and diagnostic modules of aqueous
biological molecules, since they provide great opportunities for molecular recognition
of water-soluble guests by designer hosts in an insoluble floating monolayer [3]. Enhanced binding efficiencies of host-guest recognition at the air-water interface
are in accord with theoretical simulations [4,5] and are supported experimentally as seen in selective sensing of aqueous peptides
[6-8]. We have recently applied the concept of nanotechnology to these interfacial molecular
recognition systems by embedding molecular machines in a Langmuir monolayer at the
air-water interface where their mechanical operation can be operated by compressive
surface pressure applied laterally [9]. The morphologies of the molecular machines can be controlled by macroscopic mechanical
forces, resulting in optimization of structure for molecular sensing. We have previously
demonstrated the (i) capture and release of fluorescent molecules upon cavity closure-opening
motions of molecular machines [10-13], (ii) control of enantioselective binding of amino acids upon twisting motion of
molecular machines [14,15], and (iii) discrimination of single-methyl-group difference between nucleobases (thymine
and uracil) by control of macroscopic lateral pressures [16]. In our next demonstration of the utility of host molecules at the air-water interface,
we show discrimination of some naturally occurring nucleotides, which are important
in biological activities such as energy storage and signal transduction, using cholesterol-armed-triazacyclononane
(1) as a molecular machine (see Figure 1 for recognition system). Using this strategy, we were able to discriminate between
several ribonucleotides based on the twofold to tenfold difference in their binding
constants under optimized conditions.

Experimental

Water used for the subphase was distilled using an Autostill WG220 (Yamato) and deionized
using a Milli-Q Lab (Millipore). Its specific resistance was greater than 18 MΩ ·
cm. Spectroscopic grade chloroform (Wako Pure Chemical Co., Osaka, Japan) was used
as the spreading solvent. Ribonucleotides [adenosine 5'-monophosphate disodium salt
(AMP), cytidine 5'-monophosphate disodium salt (CMP), guanosine 5'-monophosphate disodium
salt (GMP), and uridine 5'-monophosphate disodium salt (UMP)] and lithium chloride
were purchased from Wako Pure Chemical Co. (Osaka, Japan). The synthesis of the molecular
machine, cholesterol-armed-triazacyclononane (1), was described previously [16]. Isotherms of surface pressure and molecular area (π-A isotherm) were measured at 20.0°C using an FSD-300 computer-controlled film balance
(USI System, Fukuoka, Japan). A period of 15 min was allowed for spreading solvent
evaporation, compression was commenced at a rate of 0.2 mm s-1. Fluctuation of the subphase temperature was within ± 0.2°C.

Results and discussion

π-A isotherms of the molecular machine 1 with four different ribonucleotides (AMP, CMP, GMP, and UMP) in the subphase are shown
in Figure 2 (on pure water) and Figure 3 (on aqueous solution of [LiCl] = 10 mM). In general, isotherms of 1 under each condition exhibit monotonic increases without phase transitions. Increase
in the nucleotide concentration in the subphase shifted the isotherms to larger molecular
areas, suggesting that the molecular packing of 1 was disturbed by interaction between the nucleotides and 1 at the air-water interface. According to a reported method [14,16], the shifts in molecular areas at various guest concentrations can be converted into
the binding constants (K) of nucleotides to the monolayer of 1 at each surface pressure. The calculated values are summarized in Figure 4. In all the cases, assumption of an equimolecular binding gave the best fitting of
the binding curves.

Figure 4.Binding constant (K) of AMP, CMP, GMP, and UMP to the monolayer of 1 at various surface pressures at
20°C: (A) without LiCl and (B) with 10 mM of LiCl.

As shown in Figure 4A, the binding constants of the nucleotides to the monolayer of 1 gradually decreased as the surface pressure increased. This is because expansion of
the molecular area of 1 by binding to the nucleotides is thermodynamically unfavourable at higher pressures.
As will be described later, when the triazacyclononane moiety is not complexed with
a central Li+ ion electrostatic interaction between 1 and the phosphate group within the nucleotide becomes less important. Hence, on the
surface of pure water, there exists a rather ambiguous interaction between 1 and the base portion of the nucleotides, and this interaction is quite sensitive to
other factors. Although the absolute value of binding constants decreased drastically,
differences in the binding efficiencies amongst the nucleotides became obvious at
higher surface pressures. For example, ratios of binding constants, K(AMP/UMP), K(CMP/UMP), and K(GMP/UMP), are 0.78, 1.05, and 0.68, respectively, at a surface pressure of 5 mN m-1, whereas K(AMP/UMP), K(CMP/UMP), and K(GMP/UMP) values become 9.89, 8.77, and 5.52, respectively, when compressed to 35
mN m-1. Thus, discrimination of GMP and UMP from AMP and CMP is possible as well as between
GMP and UMP, although differentiation between AMP and CMP is rather difficult even
at greater surface pressures.

Complexation of Li+ ion by the triazacyclononane ring causes two variations in the characteristics of
the recognition system. The presence of Li+ ion at the core of 1 ensures strong electrostatic interaction between the monolayer and the nucleotides.
In addition, the complexation of Li+ ion stabilizes the conformation of the cyclononane ring of 1, resulting in a rather simple situation of discrimination amongst the nucleotides
(Figure 4B). Although the binding constants of UMP to the monolayer exhibit a distinct dependence
on surface pressure, an order of binding constant (K(CMP) > K(GMP) > K (AMP)) is maintained over the entire pressure range. An apparent advantage in the
Li+-containing system is due to a significant increase in the binding constant of CMP.
As seen in Figure 3B, binding of CMP to the monolayer of 1 does not require large expansion of the monolayer in contrast to AMP and GMP (Figure
3A, C). This binding mode should provide more favourable binding to the molecular assembly.
On the other hand, the binding curve for UMP is unusual when compared with the other
nucleotides. As has been suggested in previous research [16], the uridine moiety of UMP probably interacts with the cyclononane ring, thus competing
with the major interactions between the phosphate and the Li+ ion.

Conclusions

Prior to this and our other preliminary reports, discrimination of nucleotides has
not been easy to achieve because of their structural similarity, and despite its importance
in biological and pharmaceutical fields. This research strikingly demonstrates a method
for molecular discrimination amongst structurally similar nucleotides by mechanical
tuning of a simple host at a dynamic interfacial medium. Recognition and discrimination
of ribonucleotides can also be optimized. The concept of mechanical tuning for optimization
of molecular recognition should become a novel methodology in bio-related nanotechnology
as an alternative to traditional strategies based on increasingly complex and inconvenient
molecular design strategies.

Acknowledgements

This work was partly supported by World Premier International Research Center Initiative
(WPI Initiative), MEXT, Japan and Core Research for Evolutional Science and Technology
(CREST) program of Japan Science and Technology Agency (JST), Japan.